1. Field of the Invention
This invention relates generally to methods and apparatus for visually distinguishing color values, and particularly to such a method, and apparatus useful therein, for enabling more accurate results to be obtained from visual observation of color responses produced by colorimetric analytical tests such as those used to detect the presence and/or amount of various substances in test samples.
2. Background Art
Many analytical methods and devices are presently available which rely upon a visually detectable response to an analyte in a test sample, such as a color change, as a means for determining, on a semiquantitative or quantitative basis, the presence and/or concentration of an analyte in a test sample. Whether the sample being assayed is a body fluid such as urine, blood, gastrointestinal contents, spinal fluid or the like, or an industrial chemical, waste water, swimming pool water or any of a number of types of media which can be tested to determine various substances therein, it is generally considered necessary that satisfactory visual references be available against which the color or other response produced by the test means can be compared in order to provide a degree of accuracy in quantitation of the reading of the color response of the test used.
Many conventional colorimetric assay methods employ as such references standardized color charts, for visual readings, or standard reference data stored in a reflectance instrument against which the instrument compares readings taken from light reflected from color response areas of the test means which have been exposed to the analyte. In the case of the most convenient test formats now commonly used by clinical assayists--the impregnated fiber matrix, solid phase reagent test strip--the extent of reaction, and hence the concentration of an analyte in a test sample, can be correlated with the intensity, and particularly with the wavelength (hue), of light reflected from the reacted matrix after contact of the sample therewith. To visually assess, quantitatively or semiquantitatively, the amount of analyte present, the color of the reacted matrix is usually compared with predetermined color blocks of differing hues which correspond to various concentration levels of the analyte in the sample.
Thus, visual measurement of the color response of a reacted solid phase reagent test device usually involves comparison and matching of its colored response area with the predetermined colors on a scale of a standard reference chart. Typically, such color charts are made up of a number of discretely hued color blocks, each of which corresponds to a substantially different concentration level of the analyte in a test sample. Ordinarily, the blocks are set against, and surrounded by, a solid white background. By matching the color of the reacted test matrix to that of a particular block or blocks of the chart corresponding to various concentration levels and judged to be most closely related in hue, or by interpolating the matrix color between that of two or more adjacent blocks, the concentration of the analyte can be derived.
Clearly, the accuracy of the visually read results obtained by the foregoing methodology depends on the ease and certainty by which a human observer is able to discriminate between differing, but often closely related, hues of the component color blocks of the chart. Subtle differences in hue of the blocks on such charts often correspond to large quantitative differences in analyte concentration. In order to optimize visual color discrimination, it is apparent that perceived visual differences between the color of adjacent blocks, ideally, should be maximized so that, from a visual standpoint, adjacent blocks which are observed in the same field of vision are as different in hue and intensity of color as possible to the human eye. Unfortunately, because of the reagent systems used in many colorimetric analytical tests, dramatic hue differentiation between adjacent blocks on a color comparison chart is not always feasible, since, in practice, small differences in the hues produced in the reacted test matrix often correspond to very large differences in analyte concentration.
Accordingly, in order to achieve the optimum degree of quantitation available for visual readings of solid phase reagent test devices, the chemical reaction taking place in the test matrix after contact with an analyte in a sample should produce distinctly-colored reaction products, the spectral characteristics of which should vary dramatically with variances in the analyte concentration of the sample. Attempts have been made to achieve this goal to some extent in solid phase devices by using various combinations of different chromogenic reagents in the test device, or by the inclusion of background dyes in the device which interact visually with the product chromophores to achieve a desired effect for a particular analyte concentration level. Traditional solution based wet chemical analytical procedures, while ordinarily capable of somewhat better colorimetric quantitation because the reagents need not be reduced to a solid state and combined in a singular test matrix, nevertheless suffer from the aforedescribed inherent inaccuracies in color perception by human observers, as well as the disadvantages associated with the elaborate equipment and procedures necessary to carry them out.
The best quantitation heretofore available with both solid phase and wet chemical analytical tests has been achieved when the color of the reacted matrix or solution has been measured instrumentally, rather than visually. Obviously, monitoring color changes instrumentally increases the quality of the measurement by removing the subjective component of the visual read process. However, although instrumental methods enable better quantitation, often such methods lack convenience in the field and are expensive in terms of the equipment and materials necessary to carry out the tests. Thus, it has been sought to improve the reading of colorimetric solution tests, and particularly of sold phase reagent tests, in ways such as those previously set forth in order to obtain a degree of quantitation approximating that of instrumental techniques. Making such tests acceptably accurate when visually read not only would help to alleviate the aforementioned problems, but also would make such tests more feasible for very critical uses, for example, when solution or instrumental methods are not available for diagnostic use because of remote field clinical locations or cost, but high accuracy is, nevertheless, a prerequisite. Heretofore, such attempts have fallen short of their mark in terms of enabling very high degrees of quantitation, i.e., excellent correlation with actual amounts of an analyte present in a sample, while at the same time obviating the need for elaborate solution chemistry procedures or instrumentation.
It is believed that the task of providing improved quantitation for visually read, colorimetric analytical tests has not been approached from the standpoint of providing substantial improvements in the color reference standards to which the color responses of such tests are compared. However, the art is replete with scientific literature involving discussions of various phenomena involved in the perception and discrimination of closely related lightness/darkness values of colors, such as the color values typically found in such charts, by the human eye.
Color is generally accepted to be three dimensional, having the characteristics (or color values) of hue, lightness and darkness. The literature seems to suggest that the optimum condition for determining how light (or how dark) colored areas can be achieved by setting them against a background having about the same lightness/darkness value. Likewise, the literature seems to propose that the optimum condition for hue discrimination is when a background hue is made about equal to the hues being viewed against the background. So far as is known, however, no suggestion or disclosure has been made which sets forth a general relationship between the lightness/darkness dimension and the hue dimension, so that slight differences in the latter can be more easily perceived by selection of an appropriately light or dark background against which the hues are viewed, regardless of the actual hues of the colors being viewed.
For example, D. Judd and G. Wyszecki, Color in Business, Science and Industry, 3rd ed. (John Wiley and Sons, New York), and particularly pp. 285-7, 292 and 308-9 of this reference, describe and explain certain visual effects, such as the well known "crispening effect", and chromaticity effects involved in the visual discernment of sample grays against gray backgrounds or surrounds (pp. 285-7, 292). Also disclosed by these authors are effects involved in the perceived, comparative chromaticity of adjacent vision fields of slightly differing chromaticity set against a surrounding field having a chromaticity either substantially different from, or nearly the same as, that of the fields being compared (p. 308-9). The "crispening effect" is described in this reference as relating only to gray samples on gray backgrounds, so that only the lightness/darkness dimensions (expressed as V, Munsell Value, or Y, luminescence factor) of the samples and backgrounds are involved. The general relationship derived from this discussion, in terms of lightness/darkness perception of samples against a background, is that for discernment between two nearly matching gray samples, accuracy is increased when the two samples are viewed against a background gray of nearly the same V value as that of the two samples. With respect to color matching or color discrimination, this reference discloses that the foregoing concept can be extended to visual discrimination of colored samples differing only slightly from one another in hue, i.e., the ideal background for distinguishing between two similarly colored samples set against the background is of a hue similar to that of each of the two colored samples.
Therefore, the Judd and Wyszecki reference previously discussed refers to "lightness" as indicative only of the position of a given colored sample on a gray scale running from white to black, with no consideration of the hue or chromaticity of the sample as it relates to the "lightness" parameter. Likewise, the hue characteristics are referred to in this reference as apparently observing similar visual discrimination principles, but independently of "lightness", that is, the background color need only be similar in hue to the two colors being perceived against it for optimum resolution of the actual hue of one color vis-a-vis the other. This work, therefore, does not disclose or suggest any phenomenological relationship between "lightness" of a background and the ability of a human observer to accurately discriminate between colored samples set against it which may differ only slightly in hue, but not "lightness".
Additional literature references discuss topics of relevance to the general areas of color matching and visual color discrimination. These include K. L. Kelly and D. B. Judd, Color: Universal Language and Dictionary of Names, National Bureau of Standards Special Publication 440, pp. A10-A12; I. T. Pitt and L. M. Winter, Effect of surround on perceived saturation, Journal of the Optical Society of America, Vol. 64, No. 10 (October, 1974), pp. 1328-1331; C. J. Bartleson, Changes in Color Appearance with Variations in Chromatic Adaptation, COLOR research and application, Vol. 4, No. 3 (Fall, 1979), pp. 119-138; T. S. Troscianko, Effect of Subtense and Surround Luminance on the Perception of a Coloured Field, Ibid., Vol. 2, No. 4 (Winter, 1977), pp. 153-159; and R. W. G. Hunt, The Specification of Colour Appearance. II. Effects of Changes in Viewing Conditions, Ibid., Vol. 2, No. 3 (Fall, 1977), pp. 109 et seq.
All of the references cited in the preceding paragraph disclose substantially the same phenomenon: that by altering the luminance, i.e., the intensity or lightness/darkness of a surround, the actual luminance of sample colors set against the surround which have only slightly differing luminance values can be more readily distinguished. The Kelly and Judd reference also describes variable gray backgrounds of standard ISCC-NBS Centroid color charts wherein colored samples are so affixed "that each color is seen on a background of approximately its own lightness" (p. A-10). The general conclusions and mathematical derivations presented in this literature do not suggest any solution to the problem of enhancing visual discernment between very similar actual hues of colored areas by alteration or selection of the luminance (lightness/darkness) of their surround or background.
The patent art in the general area of color matching and discrimination discloses varied approaches to discerning lightness/darkness of colors against backgrounds of various types, but also appears lacking of suggestions as to a solution for the last-mentioned problem. For example, U.S. Pat. No. 1,070,891 to Hochstetter discloses a color comparer where one foreground color is displayed adjacent to another to enable close matching of the colors, rather than providing a background to enable the foreground colors to be more readily determined.
Mooney, U.S. Pat. No. 1,389,836, discloses a colorimeter wherein colors of liquids are measured or compared. The patentee points out, on page 1, column 2, beginning at line 77, that the nature of the background or the area surrounding it affects the ease of an observer's judgments. A background which is white or neutral gray and in light intensity the same as the colors being compared is recommended. However, no detailed discussion of this system of analysis is contained in the patent.
A color card is shown by Osborne, in U.S. Pat. No. 2,074,704. The patentee seeks to bring out the full color or true color by associating or combining the colored sample or area with a black background of dull or light-absorptive finish.
An apparatus for testing used lubricating oils is shown by Franzman, U.S. Pat. No. 2,245,557. The used oil is placed on a dark background which renders the dark color of the oil much lighter in appearance than when placed on a white background. It is said that this enables various shades of dark, used oil to be more easily and quickly differentiated and the degree of variation to be more prominent.
U.S. Pat. No. 2,916,963 to Bouman discloses an apparatus for testing light discrimination, wherein light of different intensities and different color is the basis upon which the discrimination is made. Atkinson et al., U.S. Pat. No. 3,438,737 (assigned to the present assignee) discloses devices for detecting protein in fluids. In column 3, beginning at line 36, there is a discussion of the use of background coloring material in the compositions of the test devices themselves. U.S. Pat. No. 3,653,771 to Piringer discloses a means for color evaluation of a color sample, and U.S. Pat. No. 3,529,519 to Mitchell discloses an apparatus for color adjustment in photographic printing. In the latter, a background surface of neutral gray is provided for comparison purposes.
Moyer et al., U.S. Pat. No. 3,791,933, discloses a method for the rapid assay of enzyme substrates and the like. The test involves comparison between the color developed in a test spot and a conventional color chart. Furutani et al., U.S. Pat. No. 4,160,646, discloses a method for analyzing liquid specimens. The test involves obtaining corrected reflectivities of the test pieces with regard to a reference piece. Faulkner, U.S. Pat. No. 4,234,313, shows a testing composition where a colored indicator loses color in direct proportion to the amount of material being tested that is present. A comparison strip is also used in this system.
U.S. Pat. No. 4,330,299 to Cerami discloses a method for measuring the level of glucose in body fluids by placing a sample of the body fluid in contact with an indicator. A kit is also disclosed having indicator means which provides a color reaction different from that of the remaining color forming materials.
Thus, because of the aforedescribed deficiencies of the art, in developing the instant invention the task was faced of formulating a color reference for visual reading against which the hue responses of colorimetric analytical tests could be compared, and which would achieve substantially improved accuracy in quantitation over any other color reference or color comparison method heretofore known. This task was particularly complicated because most color reference charts commonly used in association with such analytical tests not only have color blocks set upon a white or nearly white background, but also have adjacent color blocks thereupon which differ only slightly in actual hue. Moreover, on most such charts nonadjacent blocks differ somewhat drastically in hue, and often in at least a portion thereof, color blocks (both adjacent and nonadjacent) are of substantially the same luminance, or lightness/darkness value. Thus, the general configuration of many such color charts comprises a substantially solid white paper substrate having printed thereupon a series of solid colored blocks of various hues ranging from light to dark luminance as read from one side of the chart to the other. An indication of corresponding concentrations of an analyte is usually printed upon the chart proximate to each color block.